N-phenylcarbazole fluorescent compound for alcohol compound labeling and synthesis method and application thereof

Abstract

The application provides a synthesis method and application of an N-phenyl carbazole fluorescent compound for alcohol compound labeling, and belongs to the technical field of fluorescent labeling of organic small-molecule alcohol compounds. The application takes N-phenyl carbazole as a fluorescent parent ring, and imidazole as a reactive group, and the chemical name is 4-(9-carbazole)-benzoylimidazole. The specific preparation method is as follows: (1) reacting carbazole with p-iodobenzoate to obtain 4-(9-carbazolyl)-benzoic acid methyl ester a; (2) reacting a with a potassium hydroxide aqueous solution to obtain 4-(9-carbazolyl)-benzoic acid b; and (3) reacting b with N,N'-carbonyldiimidazole to obtain a target product. The fluorescent labeling reagent can accurately and sensitively label twelve kinds of fatty alcohols, so that the separation and detection of trace fatty alcohols in a complex system can be realized, and the fluorescent labeling and analysis of various fatty alcohols in the research fields of life and food can be carried out, and the application prospect is wide.

Description

Technical Field

[0001] This invention relates to the field of fluorescent labeling technology for small organic molecule alcohols, specifically providing an N-phenylcarbazole fluorescent compound for labeling alcohols, its synthesis method, and its application. Background Technology

[0002] Fatty alcohols are essential components of living organisms. Rapid and accurate determination of fatty alcohols is of great significance in biology, food science, and clinical medicine. It is well known that most fatty alcohols exhibit neither ultraviolet absorption nor fluorescence, making their detection at trace levels quite difficult. Currently, the search for highly sensitive determination methods using pre- or post-column derivatization with automated analyzers and novel fluorescent reagents remains a cutting-edge research area. Several photometric derivatization reagents for fatty alcohol determination suffer from low sensitivity and long derivatization times, limiting their application. Therefore, developing a novel, highly sensitive fluorescently labeled reagent for the fluorescent detection of fatty alcohols is of great importance. Summary of the Invention

[0003] To address the aforementioned problems in the existing technology, this invention provides an N-phenylcarbazole compound for fluorescent labeling of alcohol compounds, its synthesis method, and its application, which has advantages such as fast reaction rate, strong labeling product signal, high accuracy, and high sensitivity.

[0004] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0005] On one hand, this invention provides a labeling reagent for 4-(9-carbazole)-benzoic acid compounds, using 4-(9-carbazole)-benzoic acid as the fluorescent parent ring and imidazole as the reactive group. Its chemical name is 4-(9-carbazole)-benzoylimidazolium, and its chemical structural formula is as follows:

[0006]

[0007] This invention also includes a method for synthesizing an N-phenylcarbazole fluorescent compound labeling reagent, comprising:

[0008] Step 1: Carbazole is subjected to a substitution reaction with methyl p-iodobenzoate to obtain intermediate methyl 4-(9-carbazole)-benzoate.

[0009] Step 2: The intermediate methyl 4-(9-carbazolyl)-benzoate was reacted with an aqueous solution of potassium hydroxide, and then acidified to obtain the intermediate 4-(9-carbazolyl)-benzoic acid.

[0010] Step 3: React the intermediate 4-(9-carbazole)-benzoic acid with N,N'-carbonyldiimidazole to obtain the target product 4-(9-carbazole)-benzoylimidazole.

[0011] Furthermore, the synthesis method specifically includes the following steps:

[0012] Step 1: Substitution reaction: Carbazole, potassium carbonate, cuprous iodide, copper powder, and di-tert-valeryl methane were dissolved in dimethyl sulfoxide. A dimethyl sulfoxide solution containing methyl p-iodobenzoate was added dropwise under constant pressure. The reaction was carried out in an oil bath at 130°C for 45 min. After the reaction was completed, the insoluble matter was filtered off after the reaction solution was cooled. 400 mL of water was added and stirred continuously. A white solid precipitated. The solid was filtered and washed twice with water. The crude product was dried to obtain the intermediate methyl 4-(9-carbazolyl)-benzoate.

[0013] Step 2: Hydrolysis reaction: Dissolve the crude methyl 4-(9-carbazolyl)-benzoate in ethanol, add potassium hydroxide aqueous solution, react in an oil bath at 80℃ for 1 hour, cool to room temperature after the reaction is completed, add water and hydrochloric acid to precipitate, filter, take the precipitate and add sodium hydroxide aqueous solution to adjust to neutral, add hydrochloric acid to the supernatant until strongly acidic (pH 2-4), repeat this separation step, and then recrystallize twice with ethanol to obtain white needle-like crystal product.

[0014] Step 3: Substitution reaction: Dissolve the intermediate product 4-(9-carbazolyl)-benzoic acid in tetrahydrofuran, add a tetrahydrofuran solution containing N,N'-carbonyldiimidazole under electromagnetic stirring, react in an oil bath at 50°C for 30 min, and allow to stand to precipitate a white crystalline product.

[0015] Furthermore, in step 1, the molar ratio of carbazole to methyl p-iodobenzoate is preferably 1:1.2.

[0016] Furthermore, in step 3, the target product 4-(9-carbazole)-benzoylimidazolium tetrahydrofuran is recrystallized twice to obtain a white needle-like crystalline product; the molar ratio of 4-(9-carbazole)-benzoic acid to CDI is preferably 1:1.

[0017] The present invention also includes the synthesis and application of an N-phenylcarbazole fluorescent compound reagent for labeling alcohols, wherein the labeling reagent has the above-described structure or is synthesized by the above-described synthesis method, and is used to detect the content of fatty alcohols in a sample.

[0018] Furthermore, the specific steps for detecting the fatty alcohol content in the sample are as follows:

[0019] (1) Solution preparation: Prepare a 40 g / L DMAP (4-dimethylaminopyridine) aqueous solution; prepare 5 mixed fatty alcohol standard solutions with concentrations of 10 nM, 100 nM, 1 μM, 10 μM and 100 μM respectively; prepare an acetonitrile solution of 4-(9-carbazole)-benzoylimidazolium with a concentration of 10 mM;

[0020] (2) Plotting the working curve: 40 μL of DMAP solution, 20 μL of fatty alcohol, 20 μL of LDMSO mixed standard solution and 100 μL of acetonitrile solution of 4-(9-carbazole)-benzoylimidazolium were added sequentially to a 2 mL ampoule. After reacting at 80 °C for 30 min, the derivatized solution was treated as follows:

[0021] (a) Add 50 μL of quenching agent acetic acid to stop the reaction, let stand for 5-10 min and then inject the sample for analysis;

[0022] (b) Take 10 μL of the reaction solution for high performance liquid chromatography-fluorescence analysis to obtain the chromatogram of the fatty alcohol mixed standard. After derivatizing and performing high performance liquid chromatography-fluorescence analysis on the five fatty alcohol mixed standards in sequence, plot the fatty alcohol concentration working curve with fatty alcohol concentration as the abscissa and peak height as the ordinate.

[0023] (3) Detection of fatty alcohol concentration: After labeling the sample with 4-(9-carbazole)-benzoylimidazolium derivative, high performance liquid chromatography-fluorescence analysis is performed. The obtained peak area is substituted into the fatty alcohol concentration working curve to obtain the concentration of fatty alcohol in the sample.

[0024] Furthermore, the steps for testing the fatty alcohol content in hand sanitizer are as follows:

[0025] Take 5 mL of hand sanitizer, adjust it to acidic with hydrochloric acid, add 10 mL of chloroform, sonicate for 3 min, centrifuge for 10 min, separate the organic phase, wash the organic phase repeatedly with 6 mol / L HCl several times until a large amount of white precipitate no longer precipitates, blow the white precipitate dry with N2, add acetonitrile to redissolve it for later use, label it according to the method in step (2), and perform high performance liquid chromatography-fluorescence analysis. Substitute the obtained peak height into the working curve to obtain the concentration of each fatty alcohol in the hand sanitizer, and thus obtain the content of each fatty alcohol in the hand sanitizer.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The reagent of this invention has a maximum excitation wavelength of 290 nm and an emission wavelength of 443 nm, which can realize rapid fluorescent labeling and detection of fatty alcohol compounds. The reagent uses an N-phenylcarbazole ring as the fluorescent parent ring and imidazole as the reactive group. Its synthesis steps are simple and convenient and easy to realize. It can be completed in three steps: (1) Carbazole is reacted with methyl p-iodobenzoate to obtain the intermediate methyl 4-(9-carbazole)-benzoate; (2) methyl 4-(9-carbazole)-benzoate is reacted with potassium hydroxide aqueous solution to obtain the intermediate 4-(9-carbazole)-benzoic acid. After recrystallization twice with ethanol, a white needle-like crystal product 4-(9-carbazole)-benzoic acid is obtained; (3) 4-(9-carbazole)-benzoic acid is reacted with CDI to obtain the target product 4-(9-carbazole)-benzoylimidazolium. After recrystallization twice with tetrahydrofuran, a white crystalline product is obtained.

[0028] The fluorescent labeling reagent of this invention is chemically stable with a purity of 99.5%. This N-phenylcarbazole fluorescent labeling reagent uses N-phenylcarbazole as the parent ring and imidazole as the active group. The imidazole group exhibits strong selectivity for alcohols (such as fatty alcohols) when labeling them, with short labeling time and high labeling yield. Regarding derivatization conditions, the fatty alcohol fluorescent labeling reagent only requires a reaction time of 30 min at 80°C to achieve complete derivatization, without the need for complex extraction conditions. For detection, high-performance liquid chromatography (HPLC) fluorescence detection is employed.

[0029] The N-phenylcarbazole fluorescent labeling reagent of the present invention can rapidly, accurately, and sensitively label fatty alcohols, thereby realizing the separation and detection of trace amounts of fatty alcohols in complex systems. It can be used for the fluorescent labeling and analysis of various fatty alcohols in research fields such as life analysis and food analysis, and has broad application prospects. Attached Figure Description

[0030] Figure 1 This is the synthetic route diagram for the preparation of 4-(9-carbazole)-benzoylimidazolium in Example 1 of the present invention;

[0031] Figure 2 This is the mass spectrum of 4-(9-carbazole)-benzoylimidazolium prepared in Example 1 of this invention;

[0032] Figure 3 This is the UV-Vis absorption spectrum of the labeling reagent and the ethanol-labeled product in Example 1 of this invention;

[0033] Figure 4 This is the fluorescence emission spectrum of the labeling reagent and the ethanol-labeled product in Example 1 of the present invention;

[0034] Figure 5 This is a reaction route diagram of the labeling reagent and fatty alcohol in Example 2 of the present invention;

[0035] Figure 6 This is the chromatographic separation diagram of the 12 fatty alcohol mixed standards derived from the labeling reagent in Example 2 of the present invention;

[0036] Figure 7 This is the chromatogram of fatty alcohols in a real sample of hand sanitizer detected by the labeling reagent in Example 3 of the present invention. Detailed Implementation

[0037] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with specific embodiments.

[0038] Unless otherwise specified, all reagents and materials used in the examples and comparative examples are commercially available.

[0039] This invention provides a method for synthesizing N-phenylcarbazole fluorescent compounds for labeling alcohol compounds, and specific embodiments are as follows.

[0040] Example 1

[0041] An N-phenylcarbazole fluorescent compound for labeling alcohols, with N-phenylcarbazole as the fluorescent parent ring and imidazole as the reactive group, is chemically named 4-(9-carbazole)-benzoylimidazolium, and its chemical structural formula is as follows:

[0042]

[0043] For the specific synthetic route, see Figure 1 ,include:

[0044] (1) Substitution reaction: Carbazole (6.4g), potassium carbonate (5.3g), cuprous iodide (0.5g), copper powder (0.5g), and ditert-valeryl methane (3mL) were placed in dimethyl sulfoxide (50mL). A dimethyl sulfoxide solution (20mL) containing methyl p-iodobenzoate (12.04g) was added dropwise under constant pressure. The reaction was carried out in an oil bath at 130℃ for 45min. After the reaction was completed, the insoluble matter was filtered off after the reaction solution was cooled. 400mL of water was poured in and stirred continuously. A white solid precipitated. The solid was filtered and washed twice with water. The crude product was dried to obtain the intermediate methyl 4-(9-carbazolyl)-benzoate.

[0045] (2) Hydrolysis reaction: 5.0 g of crude methyl 4-(9-carbazolyl)-benzoate was dissolved in 100 mL of ethanol and 30 mL of potassium hydroxide (5.0 g) aqueous solution was added. The mixture was reacted in an oil bath at 80 °C for 1 h. After the reaction was completed, the mixture was cooled to room temperature, water was added to precipitate the product, and hydrochloric acid was added to adjust the pH to acidity. The product was filtered, and the precipitate was taken and sodium hydroxide aqueous solution was added to adjust the pH to neutral. Hydrochloric acid was added to the supernatant to make it strongly acidic. This separation step was repeated. The precipitate was then recrystallized twice with ethanol to obtain a white needle-like crystalline product.

[0046] (3) Substitution reaction: Dissolve the intermediate product 4-(9-carbazolyl)-benzoic acid (2.0 g) in tetrahydrofuran (5 mL), add a tetrahydrofuran solution (2 mL) containing N,N'-carbonyldiimidazole (1.14 g) under electromagnetic stirring, react in an oil bath at 50 °C for 30 min, and let stand to precipitate a white crystalline product.

[0047] Figure 2 The mass spectrum of the prepared white crystalline product is shown below, along with its characterization data: MS: m / z [M+H] + 338.1, demonstrating the successful preparation of 4-(9-carbazole)-benzoylimidazolium.

[0048] To improve the detection sensitivity of fluorescently labeled fatty alcohols, accurately obtaining the maximum excitation and emission wavelengths of the fluorescent labeling reagent is crucial. To obtain the maximum excitation and emission wavelengths of the labeling reagent or product, this invention labels an ethanol standard solution with a 1 μM 4-(9-carbazole)-benzoylimidazolium solution, followed by fluorescence analysis. Figure 3 It is the fluorescence excitation spectrum of the labeling reagent and the ethanol-labeled product; Figure 4 This is the fluorescence emission spectrum of the labeling reagent and the ethanol-labeled product. Figure 3-4 It can be seen that the maximum excitation wavelength and the maximum emission wavelength of the labeled product are 290 nm and 443 nm, respectively.

[0049] Example 2

[0050] This invention also provides an application of N-phenylcarbazole fluorescent compounds for labeling alcohols, used to detect the content of fatty alcohols in a sample, specifically:

[0051] (1) Prepare 40 g / L DMAP solution; prepare 5 mixed (C1-C12) fatty alcohol standard solutions, wherein the concentration of each fatty alcohol is 10 nM; prepare 10 nM mixed fatty alcohol standard solutions, and then prepare 100 nM, 1 μM, 10 μM and 100 μM solutions in sequence; prepare acetonitrile solution of 4-(9-carbazole)-benzoylimidazolium with a concentration of 10 mM.

[0052] (2) Add 40 μL of DMAP solution, 20 μL of fatty alcohol mixed standard solution, 20 μL of LDMSO and 100 μL of acetonitrile solution of 4-(9-carbazole)-benzoylimidazolium to a 2 mL ampoule in sequence. After reacting at 80 °C for 30 min, the derivatized solution is treated as follows:

[0053] (a) Add 50 μL of quenching agent acetic acid to stop the reaction, let stand for 5-10 minutes, and then inject the sample for analysis;

[0054] (b) Take 10 μL of the reaction solution for high performance liquid chromatography-fluorescence analysis to obtain the chromatogram of the fatty alcohol mixed standard. After derivatizing and performing high performance liquid chromatography-fluorescence analysis on the five fatty alcohol mixed standards, a working curve of fatty alcohol concentration is plotted with fatty alcohol concentration as the abscissa and peak area as the ordinate, as shown in Table 1.

[0055] (3) After labeling the sample with 4-(9-carbazole)-benzoylimidazolium derivative, perform high performance liquid chromatography-fluorescence analysis. Substitute the obtained peak area into the fatty alcohol concentration working curve to obtain the concentration of fatty alcohol in the sample.

[0056] Table 1

[0057]

[0058] Note: Y is the peak area, and X is the concentration.

[0059] As shown in Table 1, the limits of detection and quantitation were 0.99-1.51 μg / kg and 3.32-5.23 μg / kg, respectively, with a correlation coefficient (R0). 2 The result is greater than 0.9992, indicating that the analyte has good linearity, with intra-day and inter-day precision of 1.5-3.94% and 2.1-6.14%, respectively. This means the analytical method has good linearity, high precision, and low detection limits and quantitation limits.

[0060] The reaction route diagram of 4-(9-carbazole)-benzoylimidazolium with fatty alcohols in this invention is shown below. Figure 5 The chromatogram of the 12 fatty alcohols mixed standards after labeling and derivatization is shown below. Figure 6 .Depend on Figure 6 It is known that the N-phenylcarbazole fluorescent labeling reagent of the present invention can rapidly, accurately and sensitively label fatty alcohols, and is especially suitable for the separation and detection of trace amounts of small molecule alcohols in complex systems.

[0061] Example 3

[0062] A method for detecting the fatty alcohol content in hand sanitizer, comprising the following steps:

[0063] (1) Measure 5 mL of hand sanitizer, adjust it to acidic with hydrochloric acid, add 10 mL of chloroform and sonicate for 3 min, centrifuge for 10 min to separate the organic phase, wash the organic phase repeatedly with 6 mol / L HCl several times until a large amount of white precipitate no longer precipitates, blow the white precipitate dry with N2 and redissolve it with acetonitrile to obtain the sample to be tested.

[0064] (2) Add 40 μL of DMAP solution, 20 μL of the above-mentioned sample to be tested, 20 μL of DMSO and 100 μL of acetonitrile solution of 4-(9-carbazole)-benzoylimidazolium to a 2 mL ampoule in sequence. After reacting at 80 °C for 30 min, the derivatized solution is treated as follows:

[0065] (a) Add 50 μL of quenching agent acetic acid to stop the reaction, let stand for 5-10 minutes, and then inject the sample for analysis;

[0066] (b) 10 μL of the reaction solution was subjected to high-performance liquid chromatography-fluorescence analysis to obtain the chromatogram of the mixed fatty alcohols, see [reference needed]. Figure 7 By substituting the obtained peak area into the corresponding working curve, the concentration of each fatty alcohol in the laundry detergent can be obtained, and thus the content of each fatty alcohol in the hand soap can be obtained, as shown in Table 2.

[0067] Table 2

[0068]

[0069] As shown in Table 2, the hand sanitizer selected in this embodiment contains only propanol, hexanol, heptanol, octanol, nonanol, and dodecanol, and their concentrations were accurately measured.

[0070] Example 4

[0071] Under the same elution conditions, twelve small-molecule fatty alcohols were analyzed in triplicate. Retention time and peak area reproducibility were calculated. Recovery rates were assessed by adding three levels of fatty alcohol standard solutions to the hand sanitizer samples. Specifically:

[0072] (1) A hand sanitizer sample was obtained according to Example 3, and the concentrations of various fatty alcohols c1 in the hand sanitizer were detected;

[0073] (2) Add 40 μL of DMAP solution, 20 μL of the above-mentioned test sample, 20 μL of DMSO, and 100 μL of acetonitrile solution of 4-(9-carbazole)-benzoylimidazolium to a 2 mL ampoule. Then add mixed fatty alcohol standard samples with concentrations of 5 mg / L (5 mg / L for each of the C1-C12 fatty alcohols), 10 mg / L (10 mg / L for each of the C1-C12 fatty alcohols), and 15 mg / L (15 mg / L for each of the C1-C12 fatty alcohols), respectively. After reacting at 80 °C for 30 min, the derivatized solution is treated as follows:

[0074] (a) Add 50 μL of quenching agent acetic acid to stop the reaction, let stand for 5-10 minutes, and then inject the sample for analysis;

[0075] (b) Take 10 μL of the reaction solution for high performance liquid chromatography-fluorescence analysis to obtain the chromatogram of the mixed fatty alcohols. Substitute the obtained peak areas into the corresponding working curve to calculate the concentration c2 of each fatty alcohol. Calculate the recovery rate according to the following formula. The results are shown in Table 3.

[0076] Recovery rate = (c2-c1)×100% / concentration of mixed fatty alcohol standard sample.

[0077] Table 3

[0078]

[0079] As shown in Table 3, under the same elution conditions, the recovery rates of the twelve small molecule fatty alcohols were 88-99%, which is good, indicating that the accuracy of this method is good.

[0080] In summary, the N-phenylcarbazole compounds prepared by this invention can rapidly, accurately, and sensitively label fatty alcohols, thereby enabling the separation and detection of trace amounts of fatty alcohols in complex systems. They can be used for the fluorescent labeling and analysis of various fatty alcohols in research fields such as life analysis and food analysis, and have broad application prospects.

[0081] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of an N-phenylcarbazole fluorescent compound for labeling an alcohol compound, characterized in that, The N-phenyl carbazole fluorescent compound has a chemical structural formula as shown in the following: ； The alcohol compound is a fatty alcohol, and the application of the N-phenyl carbazole fluorescent compound is for detecting the content of fatty alcohol in a sample.

2. The use of the N-phenylcarbazole-based fluorescent compound for labeling an alcohol compound according to claim 1, characterized by, The specific steps for detecting the content of fatty alcohol in the sample are as follows: (1) Solution preparation: prepare 40 g / L 4-dimethylaminopyridine aqueous solution; prepare five mixed fatty alcohol mixed standard solution, and the concentrations are 10 nM, 100 nM, 1 μM, 10 μM and 100 μM respectively; prepare 10 mM 4-(9-carbazole)-benzoyl imidazole acetonitrile solution; (2) Working curve drawing: add 40 μL DMAP solution, 20 μL fatty alcohol mixed standard solution, 20 μL dimethyl sulfoxide and 100 μL 4-(9-carbazole)-benzoyl imidazole acetonitrile solution into a 2 mL ampoule in turn, and after reaction at 80 ℃ for 30 min, the derivatization solution is treated as follows: (a) stop the reaction by adding 50 μL quenching agent acetic acid, and stand for 5-10 min for sample analysis; (b) take 10 μL reaction solution for high performance liquid chromatography-fluorescence analysis, obtain the chromatographic separation diagram of the fatty alcohol mixed standard, and after derivatization and high performance liquid chromatography-fluorescence analysis of the five fatty alcohol mixed standards in turn, draw the working curve of the fatty alcohol concentration with the fatty alcohol concentration as the abscissa and the peak area as the ordinate; (3) Fatty alcohol concentration detection: after the sample to be detected is labeled by 4-(9-carbazole)-benzoyl imidazole according to step (2), high performance liquid chromatography-fluorescence analysis is performed, the obtained peak area is substituted into the working curve of the fatty alcohol concentration, and the concentration of the fatty alcohol in the sample can be obtained.

3. The use of the N-phenylcarbazole-based fluorescent compound for labeling an alcohol compound according to claim 2, characterized by, In step (1), the mixed fatty alcohol is methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, nonanol, decanol, undecanol and dodecanol.

4. The use of the N-phenylcarbazole-based fluorescent compound for labeling an alcohol compound according to claim 2, characterized by, The application is for detecting the content of fatty alcohol in hand sanitizer, and the specific steps are as follows: Take 5 mL hand sanitizer, adjust to acidity with hydrochloric acid, add 10 mL chloroform, ultrasonic oscillation for 3 min, centrifugal for 10 min, separate the organic phase, wash the organic phase with 6 mol / L HCl repeatedly for several times until a large amount of white precipitate no longer precipitates, dry the white precipitate with N2, and then redissolve in acetonitrile, label according to the method of step (2) of claim 2, and then perform high performance liquid chromatography-fluorescence analysis, substitute the obtained peak area into the working curve, and then the concentration of each fatty alcohol in the hand sanitizer can be obtained, and the content of each fatty alcohol in the hand sanitizer can be obtained.

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